Supplementary Materials for

Enhancing natriuretic peptide signaling in adipose tissue, but not in

muscle, protects against diet-induced obesity and insulin resistance

Wei Wu, Fubiao Shi, Dianxin Liu, Ryan P. Ceddia, Robert Gaffin, Wan Wei,

Huafeng Fang, E. Douglas Lewandowski, Sheila Collins*

*Corresponding author. Email: scollins@sbpdiscovery.org

Published 25 July 2017, Sci. Signal. 10, eaam6870 (2017)

DOI: 10.1126/scisignal.aam6870

This PDF file includes:

Fig. S1. Blood pressures and circulating NP concentrations of Nprc−/−, NprcMKO,

and NprcAKO mice.

Fig. S2. Expression of Nprc and Npra in skeletal muscle and adipose tissue.

Fig. S3. Alternative representation of CLAMS indirect calorimetry data of HFD-

fed NprcMKO and NprcAKO mice.

Fig. S4. HFD-fed NprcAKO and Nprcfl/fl mice show comparable expression of

thermogenic-related genes in gWAT and iWAT.

Fig. S5. Adipocyte size distribution in iWAT and gWAT of HFD-fed Nprcfl/fl and

NprcAKO mice.

Fig. S6. NPRC deficiency enhances NP signaling.

Table S1. PCR primer sequences.

www.sciencesignaling.org/cgi/content/full/10/489/eaam6870/DC1

Fig. S1. Blood pressures and circulating NP concentrations of Nprc−/−, NprcMKO, and

NprcAKO mice. (A to C) Circulating ANP (left) and BNP (right) concentrations in (A) Nprc-/-

, (B)

NprcMKO

and (C) NprcAKO

mice (n=5 for each group). The variation of plasma NP concentration

between these three lines may be due to differences in the duration of storage in the freezer. (D)

Systolic blood pressure (SBP), diastolic blood pressure (DBP), mean artery pressure (MAP) and

heart rate (HR) of NprcAKO

and NprcMKO

mice (n=5 for each group) examined by in vivo pressure

hemodynamics as described in Materials and Methods. MAP=2/3*DBP+1/3*SBP.

Fig. S2. Expression of Nprc and Npra in skeletal muscle and adipose tissue. (A to B) qRT-

PCR for the expression of Nprc (A) and Npra (B) relative to 36B4 in gastrocnemius (GA),

tibialis anterior (TA), soleus (SO), extensor digitorum longus (EDL), iWAT, gWAT, BAT and

liver of Nprcfl/fl

mice (n=4-5). The average of Ct values for each tissue is as indicated. (C to D)

qRT-PCR for the expression of Nprc (C) and Npra (D) relative to 36B4 in quadriceps (QU),

gastrocnemius (GA), tibialis anterior (TA), soleus (SO) and extensor digitorum longus (EDL) of

NprcMKO

(n=3) and Nprcfl/fl

(n=4) mice. (E to F) qRT-PCR for the expression of Nprc (E) and

Npra (F) relative to 36B4 in iWAT, gWAT, BAT, liver and kidney of NprcAKO

(n=14) and Nprcfl/fl

(n=6) mice. (G) Western blotting analysis for NPRC on lysates from iWAT, gWAT, BAT, liver

and heart of NprcAKO

and Nprcfl/fl

mice. Blots are representative of three independent experiments.

*, p<0.05; **, p<0.01; ***, p<0.001. P-values were calculated using unpaired two-tailed

Student’s t-tests.

Fig. S3. Alternative representation of CLAMS indirect calorimetry data of HFD-fed

NprcMKO and NprcAKO mice. (A to B) O2 consumption (VO

2), CO

2 production (VCO

2) rates and

energy expenditure (EE) of NprcMKO

(n=6) and Nprcfl/fl

(n=5) mice measured by indirect

calorimetry using CLAMS after 6 weeks on HFD. Data were normalized to total body mass (A)

or per mouse (B) (C to D) O2 consumption (VO

2), CO

2 production (VCO

2) rates and energy

expenditure (EE) of NprcAKO

(n=8) and Nprcfl/fl

(n=6) mice measured by indirect calorimetry

using CLAMS after 5 weeks on HFD. Data were normalized to total body mass (C) or per mouse

(D). P values were calculated using two-way ANOVA.

Fig. S4. HFD-fed NprcAKO and Nprcfl/fl mice show comparable expression of thermogenic-

related genes in gWAT and iWAT. (A to B) qRT-PCR for the expression of genes encoding

thermogenic, mitochondrial and fatty acid oxidation (FAOx) markers in gWAT (A) and iWAT (B)

of NprcAKO

(n=14) and Nprcfl/fl

(n=7) mice after 12 weeks on HFD. *, p<0.05. P-values were

calculated using unpaired two-tailed Student’s t-tests. (C) Immunostaining of UCP1 in iWAT and

gWAT of NprcAKO

(n=2) and Nprcfl/fl

(n=2) mice after 12 weeks on HFD. Scale bar: 200μm.

Fig. S5. Adipocyte size distribution in iWAT and gWAT of HFD-fed Nprcfl/fl and NprcAKO

mice. (A to B) Average adipocyte areas (left) and the distribution of adipocyte areas (right) in (A)

iWAT and (B) gWAT. ***, p<0.001. P values were calculated using unpaired two-tailed Student’s

t-tests.

Fig. S6. NPRC deficiency enhances NP signaling. (A) Oil Red O staining of adipocytes

differentiated from the stromal vascular fraction of iWAT of NprcAKO

and Nprcfl/fl

mice. Scale bar:

100μm. iWATs from 3-5 mice of each genotype were pooled together for each experiment.

Images were representative of three independent experiments. (B) Western blot analysis of PKG

activity (p-Ser239 VASP) performed on lysates from in primary adipocytes treated with vehicle

(Veh) or ANP and BNP (200nM each, A+B) for 30 min. Blots are representative of three

independent experiments. (C) Glycerol release into the media by primary adipocytes treated with

vehicle (Veh) or ANP and BNP (200nM each, A+B) for 20 hrs. *, p<0.05; **, p<0.01; n=4

independent biological repeats for each experiment, data is representative of three independent

experiments. P values were calculated using unpaired two-tailed Student’s t-tests. These values

do not take into account the possibility of intracellular glycerol being re-esterifed by glycerol

kinase. (D) Western blot analysis of proteins involved in lipolysis (p-Ser563 HSL, and ATGL),

lipogenesis (ACC) and adipogenesis (PPARγ) performed on the lysates from primary adipocytes

treated with vehicle (Veh), ANP (200nM) or ANP and BNP (200nM each, A+B) for 20 hrs. Blots

are representative of three independent experiments. (E) cGMP dose-response of HEK293-GCA

cells transfected either with YFP or NPRC-YFP plasmid. Graph is representative (means ± SD)

of three independent experiments. (F) Western blot analysis of NPRC-YFP, YFP and NPRA with

the lysate of corresponding samples in E. Blots are representative of three independent

experiments.

Table S1. PCR primer sequences.

Genes Forward (5’-3’) Reverse (5’-3’)

36B4 GATGCCCAGGGAAGACAG ACAATGAAGCATTTTGGATAATCA

Nprc AGCTGGCTACAGCAAGAAGG CGGCGATACCTTCAAATGTC

Npra TGGAGACACAGTCAACACAGC CGAAGACAAGTGGATCCTGAG

Ucp1 GGCCTCTACGACTCAGTCCA TAAGCCGGCTGAGATCTTGT

Pgc1α GAAAGGGCCAAACAGAGAGA GTAAATCACACGGCGCTCTT

Cidea GTCTGCAAGCAACCAAAGAA ATTGAGACAGCCGAGGAAGT

Dio2 CGCTCCAAGTCCACTCGCGG CGGCCCCATCAGCGGTCTTC

Elovl3 ACTTCGAGACGTTTCAGGACTTA GACGACCACTATGAGAAATGAGC

Prdm16 CAGCACGGTGAAGCCATTC GCGTGCATCCGCTTGTG

Cpt1b GAGTGACTGGTGGGAAGAATATG GCTGCTTGCACATTTGTGTT

Cycs ACCAAATCTCCACGGTCTGTTCGG GGTGATGCCTTTGTTCTTGTTGGC

Cox7α1 CGAAGAGGGGAGGTGACTC AGCCTGGGAGACCCGTAG

Acox1 GCCCAACTGTGAC GCCAGGACTATCG

Cidec GGCTCACAGCTTGGAGGA CTCCACGATTGTGCCATCT

Accα CCACACTGAACCGGAAATCT ATTTGTCGTAGTGGCCGTTC

Fasn TTCCAAGACGAAAATGATGC AATTGTGGGATCAGGAGAGC

Scd1 CCTGCGGATCTTCCTTATCATT GATCTCGGGCCCATTCG

Srebf1 ATCCAGGTCAGCTTGTTTGCGATG TGGACTACTAGTGTTGGCCTGCTT

F4/80 CTTTGGCTATGGGCTTCCAGTC GCAAGGAGGACAGAGTTTATCGTG

Cd68 TCCAAGATCCTCCACTGTTG ATTTGAATTTGGGCTTGGAG

Tnfα ACGGCATGGATCTCAAAGAC AGATAGCAAATCGGCTGACG

Il-1β TGCAGAGTTCCCCAACTGGTACATC GTGCTGCCTAATGTCCCCTTGAATC

Arg1 CTCCAAGCCAAAGTCCTTAGAG AGGAGCTGTCATTAGGGACATC

Il-10 GCTCTTACTGACTGGCATGAG CGCAGCTCTAGGAGCATGTG

Adipoq GCAGGCATCCCAGGACATC GCGATACATATAAGCGGCTTCT

Fgf21 TGGGGGTCAAGTCCGGCAGA TCCAGGAGACTTTCTGGACTGCGG

Ngr4 TCTGTCGGCAGCTTTCGT CTGAAGGTGGCCCTTCCT

Bmp8b CTGTATGAACTCCACCAACCAC GGGGATGATATCTGGCTTCA

Cd36 TGGCCAAGCTATTGCGACAT AGGCATTGGCTGGAAGAACA

Lpl AGCAGCAAGACCTTCGTGG TCTCTCTTGTACAGGGCGGC

ND1(mt) CCCATTCGCGTTATCTT AAGTTGATCGTAACGGAAGC

LPL (nu) GGATGGACGGTAAGAGTGATTC ATCCAAGGGTAGCAGACAGGT